Cost effective, renewable and sustainable energy is a global concern, which has increased investigations into alternative fuel sources. Starch-rich biomass together with sugarcane represents the main substrates for bioethanol production (Bai et al., 2008). It is produced by plants as an energy store and consists of α-1,4 linked glucose units with α-1,6 branching points. The amylose and amylopectin polymers are densely packed in starch granules forming a semi-crystalline structure with inter- and intra-molecular bonds.
A combination of α-amylases and glucoamylases is required for the complete hydrolysis of starch. Starch granules are insoluble in cold water and are often resistant to enzymatic hydrolysis (Uthumporn et al., 2010). The conventional process for the conversion of starch to ethanol requires a heat intensive liquefaction step to gelatinise the starch and thermostable α-amylases, followed by saccharification with glucoamylases. The high temperatures required for the initial processes usually account for approximately 30-40% of the total energy required for ethanol production (Szymanowska-Powalowska et al., 2012).
An alternative to this is a cold hydrolysis process at temperatures below the onset of starch gelatinization (65° C. for corn) (Robertson et al., 2006). Benefits of this process include reduced energy requirements and a higher nutritional content for the dried distillers' grains with solubles (DDGS) (Nkomba et al., 2016). DDGS are produced in large quantities during bioethanol production and represent a valuable ingredient for livestock feed (Brehmer et al., 2008).
Consolidated bioprocessing (CBP) combines enzyme production, hydrolysis and fermentation into a one-step process for bioethanol production at low temperatures. This technology represents a promising alternative for the economic production of biofuel from lignocellulosic and starchy feedstocks. CBP could simplify operational processes (e.g. number of control steps and reaction vessels) and therefore reduce maintenance and production costs. CBP systems use a single organism that is able to produce the enzymes required for hydrolysis of starch at low temperatures, i.e. cold hydrolysis, as well as convert the resultant sugars to ethanol. The cold process requires amylases that have the ability to digest raw starch efficiently at fermentation conditions. A few raw starch hydrolyzing amylases have been reported to date (Mamo and Gessesse, 1999; Robertson et al., 2006; Celińska et al., 2015). These amylases differ from conventional amylases in their affinity and interaction with the microcrystalline structures of starch granules. A starch binding domain (SBD) is a key characteristic of these enzymes and enables them to bind effectively to the surface of raw starch granules.
A comprehensive review on consolidated bioprocessing systems by Salehi Jouzani and Taherzadeh (2015) highlighted different CBP strategies, diversity in substrate types and the organisms involved in fermenting the sugars. One of the main challenges remains the simultaneous production of the amylases with high substrate affinities and specific activity (den Haan et al., 2013). In addition, fermentation requirements are ethanol concentrations in excess of 10-12% (w·v−1) within 48 to 72 hours (Bothast and Schlicher, 2005). For example, raw starch amylase encoding genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera (Eksteen et al., 2003; Knox et al., 2004), Rhizopus arrhizus (Yang et al., 2011), Aspergillus tubingensis (Viktor et al., 2013) and Thermomyces lanuginoses and S. fibuligera or L. kononenkoae (LKA1) protein (U.S. Pat. No. 9,243,256) have been expressed in Saccharomyces cerevisiae, a yeast which is an efficient ethanol producer but which on its own lacks the ability to degrade starch.
However, none of these transformed yeasts produce sufficient amounts of amylase to support efficient conversion of raw starch to ethanol in a single step at commercial scale. Although a bioengineered S. cerevisiae strain that secretes a glucoamylase is commercially available (TransFerm® from Lallemand (www.ethanoltech.com/transferm)), it lacks the required α-amylase enzymes for starch liquefaction (den Haan et al., 2015) and is therefore only a semi-CBP yeast. The TransFerm® yeast strain is thus only suitable for the conventional (warm) process, as it only consolidates the saccharification and fermentation processes after starch liquefaction. CBP has therefore not yet been implemented on a commercial level, with the main challenge being the availability of a microorganism that can express suitable enzymes and have a high fermentation capacity.
Other cold simultaneous saccharification and fermentation (SSF) processes have been developed for ethanol production from starchy substrates (Balcerek and Pielech-Przybylska, 2013; Szymanowska-Powalowska et al., 2014; Nkomba et al., 2016). In these processes, granular starch hydrolyzing enzyme (GSHE) cocktails are added to the feedstock in addition to the yeast. Genencor's STARGEN 001™ and STARGEN 002™ cocktails (Dupont-Danisco, Itasca, Itasca) hydrolyse raw starch at low temperatures (48° C. recommended for SSF), while POET (Sioux Falls, South Dekota, USA) uses a patented blend of Novozymes enzymes (POET BPX technology) in an SSF process (Görgens et al., 2015). However, these cold starch hydrolysis processes require high enzyme loadings and the cost of the commercial enzymes, e.g. STARGEN™ (Genencor International, California, USA), is high.
There thus remains a need for a yeast which can be used in a CBP process for producing ethanol from raw starch, without requiring the addition of amylases from a source other than the yeast.